Epoxide polymer surfaces

ABSTRACT

Method and reagent composition for covalent attachment of target molecules, such as nucleic acids, onto the surface of a substrate. The reagent composition includes epoxide groups capable of covalently binding to the target molecule. Optionally, the composition can contain photoreactive groups for use in attaching the reagent composition to the surface. The reagent composition can be used to provide activated

Cross Reference To Related Applications

This application is a continuation application of application Ser. No.09/521,545 filed on Mar. 9, 2000 now U.S. Pat. No. 6,762,019, which is acontinuation-in-part of application Ser. No. 09/227,913 filed on Jan. 8,1999, now U.S. Pat. No. 6,465,178, which is a continuation-in-part ofapplication Ser. No. 08/940,213 filed on September 30, 1997, now U.S.Pat. No. 5,858,653, which application(s) are incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to reagents and support surfaces forimmobilization of biomolecules, such as nucleic acids and proteins.

BACKGROUND OF THE INVENTION

The immobilization of deoxyribonucleic acid (DNA) onto support surfaceshas become an important aspect in the development of DNA-based assaysystems, including the development of microfabricated arrays for DNAanalysis. See, for instance, “The Development of Microfabricated Arraysof DNA Sequencing and Analysis”, O'Donnell-Maloney et al., TIBTECH14:401-407 (1996). Generally, such procedures are carried out on thesurface of microwell plates, tubes, beads, microscope slides, siliconwafers or membranes.

A commonly used method for immobilizing cDNA's or PCR products intoarrays is to first coat glass slides with polylysine, then apply the DNAand illuminate with UV light to photocrosslink the DNA onto thepolylysine (for example, see Schena M, Shalon D, Heller R, Chai A, BrownP O, Davis R W, “Parallel Human Genome Analysis: Microarray-basedExpression Monitoring of 1000 Genes”, Proc. Natl. Acad. Sci. USA93(20):10614-9 (1996)). One disadvantage of this approach is that the UVcrosslinking causes undesirable damage to the DNA that is not all usefulfor the immobilization. Another disadvantages of this approach is thatUV crosslinking tends to be limited to longer nucleic acids (e.g., overabout 100-mers), as provided by cDNA's and PCR products (and in contrastto the shorter nucleic acids typically formed by synthesis and referredto as “oligonucleotides”). It appears that the potential damage inducedby UV radiation (e.g., the formation of thymine dimers) is simply toogreat, and/or the extent of immobilization is insufficient, to permitshorter nucleic acids to be used. A population of longer nucleic acids,however, even when crosslinked by UV, will typically provide ampleundamaged regions sufficient to permit accurate hybridization.

Only relatively few approaches to immobilizing DNA, to date, have foundtheir way into commercial products. One such product for immobilizingoligonucleotides onto microwell plates is known as [“NucleoLink®”]NUCLEOL™, and is available from Nalge Nunc International (see, e.g.,Nunc Tech Note Vol. 3, No. 17). In this product, the DNA is reacted witha carbodiimide to activate 5′-phosphate groups, which then react withfunctional groups on the surface. Disadvantages of this approach arethat it requires the extra step of adding the carbodiimide reagent aswell as a five hour reaction time for immobilization of DNA, and it islimited to a single type of substrate material.

As another example, Pierce has introduced a proprietary DNAimmobilization product known as [“Reacti-Bind™] REACTI-BIND^(TM) DNACoating Solutions” (see “Instructions REACTI-BIND^(TM) DNA CoatingSolution” January, 1997). This product is a solution that is mixed withDNA and applied to surfaces such as polystyrene or polypropylene. Afterovernight incubation, the solution is removed, the surface washed withbuffer and dried, after which it is ready for hybridization. Althoughthe product literature describes it as being useful for all commonplastic surfaces used in the laboratory, it does have some limitations.For example, Applicants were not able to demonstrate usefulimmobilization of DNA onto polypropylene using the manufacturer'sinstructions. Furthermore, this product requires large amounts of DNA.The instructions indicate that the DNA should be used at a concentrationbetween 0.5 and 5 μg/ml.

Corning sells a product called “DNA-BIND™” for use in attaching DNA tothe surface of a well in a microwell plate (see, e.g., the DNA-BIND™“Application Guide”). The surface of the DNA-BIND™ plate is coated withan uncharged, nonpolymeric, low molecular weight, heterobifunctionalreagent containing an N-oxysuccinimide (NOS) reactive group. This groupreacts with nucleophiles such as primary amines. The heterobifunctionalcoating reagent also contains a photochemical group and spacer arm whichcovalently links the reactive group to the surface of the polystyreneplate. Thereafter, amine-modified DNA can be covalently coupled to theNOS surface. The DNA is modified by adding a primary amine either duringthe synthesis process to the nascent oligomer or enzymatically to thepreformed sequence. Since the DNA-BIND™ product is polystyrene based, itis of limited use for those applications that require elevatedtemperatures such as thermal cycling. Corning also sells anaminosilane-coated glass slide, under the tradename CMT-GAPS™ coatedslides, which uses the same protocol as polylysine-coated slides forimmobilizing DNA into microarrays.

TeleChem International, Inc. sells slides coated with an aldehyde silaneas well as an aminosilane-coated slide. The aldehyde silane slides havevery high backgrounds when fluorescence is used for detection. They alsorequire an additional reduction step for stable immobilization.

Finally, SurModics, Inc., the assignee of the present invention, hasrecently introduced a coated glass slide, under the tradename 3D-Link™that consists of a hydrophilic polymer containing amine-reactive estergroups immobilized onto the surface. For best results, this product alsorequires amine modification of the DNA to be immobilized. As expected,however, the reactive ester groups tend to be hydrolytically unstable,which limits the amount of time arrays can be printed without some lossof performance to approximately eight hours.

The role of epoxide groups, in the course of binding or immobilizingnucleic acids, has been described in various ways as well. For instance,Shi et al., U.S. Pat. No. 5,919,626 describe the attachment ofunmodified nucleic acids to silanized solid phase surfaces. The methodinvolves the use of conventional nonpolymeric reagents such asmercapto-silanes and epoxy-silanes which bond to the surface by formingsiloxane bonds with OH groups on the glass surface.

See also, U.S. Pat. No. 5,925,552 (Keogh, et al., “Method for Attachmentof Biomolecules to Medical Devices Surfaces”), which provides a methodfor forming a coating of an immobilized biomolecule on a surface of amedical device to impart improved biocompatibility for contacting tissueand bodily fluids. One such method includes converting a biomoleculecomprising an unsubstituted amide moiety into an amine-functionalmaterial, combining the amine-functional material with a medical devicebiomaterial surface comprising a chemical moiety (such as, for example,an aldehyde moiety, an epoxide moiety, an isocyanate moiety, a phosphatemoiety, a sulphate moiety or a carboxylate moiety) which is capable offorming a chemical bond with the amine-functional material, to bond thetwo materials together to form an immobilized biomolecule on a medicaldevice biomaterial surface. Included within the long list ofbiomolecules described as being useful in this patent were “a DNAsegment, a RNA segment, a nucleic acid” and others.

Also on the subject of epoxides, Nagasawa et al., (J. Appl. Biochem.7:430-437, 1985) describe the use of Sepharoses activated withepichlorohydrin or bisoxirane (both of which provide epoxide groups) forimmobilizing DNA as immunosorbents for DNA antibodies. See also,Wheatley, et al., J. Chromatog. A 726:77-90 (1996) and Potuzak, et al.,Nucl. Acids Res. 5:297-303 (1978).

To date, however, there appears to be no description in the art, letalone commercial products, that provide an optimal combination of suchproperties as hydrolytic stability, ease of use, minimized DNA damage(due to exposure to crosslinking radiation), and the ability toimmobilize underivatized nucleic acids and/or shorter nucleic acidsegments. In turn, there appear to be no products presently available,nor descriptions in the art, that provide or suggest the ability to usepolymer-pendent epoxide groups adapted to immobilize either short orlong nucleic acids, let alone in both derivatized and underivatizedforms, and suitable for immobilization onto surfaces.

Finally, Surmodics, Inc., the assignee of the present invention, haspreviously described a variety of applications for the use ofphotochemistry, and in particular, photoreactive groups, e.g., forattaching polymers and other molecules to support surfaces. See, forinstance, U.S. Pat. Nos. 4,722,906, 4,979,959, 5,217,492, 5,512,329,5,563,056, 5,637,460, 5,714,360, 5,741,551, 5,744,515, 5,783,502,5,858,653, and 5,942,555.

SUMMARY OF THE INVENTION

The present invention provides a method and epoxide-based reagentcomposition for covalent attachment of target molecules onto the surfaceof a substrate, such as microscope slides, microwell plates, tubes,silicon wafers, beads or membranes. In a preferred embodiment, themethod and composition are used to immobilize nucleic acid probes ontomicroscope slides, e.g., for use in printing DNA microarrays. The methodand reagent of the present invention can be used to covalentlyimmobilize either derivatized (e.g., amine-derivatized) or underivatized(i.e., not having a group added for the purpose of thermochemicalreaction with an epoxide group) nucleic acids, and are particularlyuseful for underivatized nucleic acids. The immobilization method ofthis invention is very convenient to perform. In a preferred embodiment,the method involves the steps of coating a support with the reagent ofthis invention, printing the nucleic acid array, incubating the slide ina humid environment, blocking excess epoxide groups and washing theslide, after which it is ready for a hybridization assay.

Parent application U.S. Ser. No. 09/227,913, describes, inter alia, acomprehensive method and reagent composition for covalent attachment oftarget molecules onto the surface of a substrate, using a reagent thatcontains one or more thermochemically reactive groups (i.e., groupshaving a reaction rate dependent on temperature). Suitable groups areselected from the group consisting of activated esters such asN-oxysuccinimide (“NOS”), epoxide, azlactone, activated hydroxyl andmaleimide groups.

The present application is particularly concerned with reagents havingepoxide groups, in the manner described above, and provides furtherexamples and advantages concerning the use of such epoxide-basedreagents. Such advantages include, for instance, the ability to use thereagents to attach underivatized DNA, in addition to DNA derivatized tocontain amine or other reactive groups, as described in parentapplication U.S. Ser. No. 09/227,913. Such advantages also includeimproved resistance to hydrolysis demonstrated by epoxides, as comparedfor instance, to NOS groups.

The present invention provides a method for immobilizing biomolecules,such as biopolymers selected from nucleic acids, proteins, andpolysaccharides, the method comprising the steps of:

a) providing a solid support having a surface,

b) providing a reagent comprising one or more epoxide groups, andoptionally also comprising one or more photogroups,

c) coating the reagent on the support surface (e.g., covalentlyattaching the polymeric reagent to the support surface by activation ofthe photogroups),

d) providing a biopolymer having a corresponding thermochemical reactivegroup,

e) attaching the biopolymer to the support by reacting its correspondingreactive group with the bound epoxide group,

f) optionally blocking the remaining epoxide groups (e.g., using anamine reagent), and

g) using the resultant coated support surface for its intended purpose,e.g., for the immobilization of biomolecules such as nucleic acids.

Applicants have found that polymers (and particularly hydrophilicpolymers) containing epoxide groups, of the type described herein, haveseveral advantages for DNA immobilization over previously used methods.These polymers, when coated onto silane-modified glass slides, forinstance, provide an improved method for immobilizing underivatized DNA.Therefore, using these reagents, it is not necessary to modify the DNAwith amines or other functional groups. Furthermore, the epoxide groupsare significantly more stable to hydrolysis than are the amine-reactiveester groups. Compared with UV crosslinking of DNA onto polylysine oraminosilane, the coated surfaces of this invention are more convenientto use and tend to result in fewer undesirable side reactions, therebyresulting in less modification of the DNA.

A reagent composition of the invention preferably provides one or moreepoxide (also known as “oxirane”) groups pendent on a polymericbackbone, such as a hydrophilic polyacrylamide backbone. Optionally, andpreferably, the reagent composition can also provide one or more pendentphotoreactive groups. The photoreactive groups (alternatively referredto herein as “photogroups”) can be used, for instance, to attach reagentmolecules to the surface of the support upon the application of asuitable energy source such as light. The epoxide groups, in turn, canbe used to form covalent bonds with appropriate functional groups on thetarget molecule.

Optionally, the composition and method of this invention can be providedin the manner described in parent application U.S. Ser. No. 08/940,213.In such an embodiment, the reagent composition can be used for attachinga target molecule to the surface of a substrate, and comprises one ormore groups for attracting the target molecule to the reagent, and oneor more epoxide groups for forming covalent bonds with correspondingfunctional groups on the attracted target molecule. Optionally, such acomposition further provides photogroups for use in attaching thecomposition to a surface. In one embodiment, for instance, a pluralityof photogroups and a plurality of ionic groups (e.g., cationic groups)are attached to a hydrophilic polymer backbone. This polymer can then becoimmobilized with a second polymer backbone that provides theabove-described epoxide groups for immobilization of target molecules.Suitable ionic groups include quaternary ammonium salts, protonatedtertiary amines and other cationic groups such as phosphonium compounds.Also included are tertiary amine groups capable of being protonated whenplaced in an acid environment. Quaternary ammonium salts include alkylquaternary ammonium compounds, such as[3-(methacryloylamino)propyl]trimethylammonium chloride (MAPTAC), aswell as aromatic quaternary ammonium groups such as pyridiniumcompounds. Phosphonium compounds include polymers prepared from monomerssuch as tributyl(4-vinylbenzyl)phosphonium chloride, and are describedin J. Appl. Polymer Sci. 53:1237 (1994), the disclosure of which is alsoincorporated by reference.

The invention further provides a method of attaching a target molecule,such as a DNA molecule, to a surface, by employing a reagent asdescribed herein. In turn, the invention provides a surface havingtarget molecules such as nucleic acids attached thereto by means of sucha reagent, as well as a material (e.g., microscope slide) that providessuch a surface.

Generally, the reagent molecules will first be attached to the surfaceby activation of the photogroups, thereafter the target molecule, (e.g.,a nucleic acid) is contacted with the bound reagent under conditionssuitable to permit it to come into binding proximity with the boundpolymer. The target molecule is thermochemically coupled to the boundreagent by reaction between the reactive groups of the bound reagent andappropriate functional groups on the target molecule.

The invention further provides a method of attaching a target molecule,such as a nucleic acid, to a surface, by employing a reagent asdescribed herein. As used herein, with regard to describing the presentinvention, the word “oligonucleotide” (or “oligo”) shall refer to asynthetic nucleic acid (as opposed to enzymaticaly prepared), and onethat is typically shorter (e.g., on the order of 100-mer or less) thancDNA or PCR products formed enzymatically. The term “nucleic acid”, inturn, will refer to all such products collectively.

The invention further provides a surface having nucleic acids attachedthereto by means of such a reagent, as well as a material (e.g., a slideor microwell plate) that provides such a surface. In yet another aspect,the invention provides a composition comprising a reagent of thisinvention in combination with a target molecule that contains one ormore functional groups reactive with the thermochemically reactive groupof the reagent.

DETAILED DESCRIPTION

A preferred reagent composition of the present invention comprises ahydrophilic polymer bearing one or more pendent epoxide groups adaptedto form a covalent bond with corresponding reactive groups of a targetmolecule, and also bearing one or more pendent photoreactive groupsadapted to be used for attaching the reagent to a surface, eitherbefore, during and/or after reaction between the reagent and the targetmolecules. Optionally, a composition can include other components, inaddition to the reagent polymer component, such as polymers havingpendent ionic groups, and the like.

In another embodiment of the invention, it is possible to immobilize thereagent composition, and in turn the target molecules, without the useof the photoreactive group. For instance, the surface of the material tobe coated can be provided with thermochemically reactive groups, whichcan be used to immobilize hydrophilic polymers having epoxide groups asdescribed above. For example, a surface may be treated with an ammoniaplasma to introduce a limited number of reactive amines on the surfaceof the material. If this surface is then treated with a hydrophilicpolymer having epoxide groups, then the polymer can be immobilizedthrough reaction of the epoxide groups with amines on the surface.Preferably, the concentration of epoxide groups on the polymer is insufficient excess, relative to the concentration of amines on thesurface to insure that a sufficient number of reactive groups remainfollowing the immobilization to allow coupling with the nucleic acidsequence.

A polymeric backbone can be either synthetic or naturally occurring, andis preferably a synthetic copolymer of the epoxide monomer and diluentor other monomers resulting from addition or condensationpolymerization. Naturally occurring polymers, such as polysaccharides orpolypeptides can be used as well. Preferred diluent monomers arebiologically inert, in that they do not provide a biological functionthat is inconsistent with, or detrimental to, their use in the mannerdescribed.

Suitable diluent monomers for use in preparing a reagent of thisinvention include acrylics such as hydroxyethyl acrylate, hydroxyethylmethacrylate, glyceryl acrylate, glyceryl methacrylate; acrylamidederivatives, such as acrylamide, methacrylamide, and acryloylmorpholine. Other synthetic polymers can be synthesized to includependent epoxide groups in the manner described herein, including vinylssuch as polyvinyl pyrrolidone and polyvinyl alcohol; nylons such aspolycaprolactam, polylauryl lactam, polyhexamethylene adipamide andpolyhexamethylene dodecanediamide; polyurethanes and polyethylene oxide,as well as combinations and copolymers thereof.

A reagent of the present invention preferably includes a hydrophilicpolymer bearing a desired average number of photogroups and epoxidegroups per average unit length or molecular weight, the combinationdependent upon the reagent selected. The epoxide monomer can also beselected to provide any desirectivity. The diluent comonomers arepreferably hydrophilic (e.g., water soluble), with acrylamide andvinylpyrrolidone being particularly preferred.

Epoxide-containing polymers of the present invention can be preparedfrom monomers, such as glycidyl acrylate, glycidyl methacrylate,allylglycidyl ether, and glycidyl vinyl ether. Useful monomers areavailable from a variety of sources, for instance glycidyl acrylate(Pfaltz & Bauer Chemicals, cat. # G03480), glycidyl methacrylate(Aldrich cat # 15,123-8), allylglycidyl ether (Aldrich, cat # A3,260-8),glycidyl vinyl ether (Aldrich, cat. # 45,865-1), and glycidylvinylbenzyl ether (Aldrich cat. # 45,867-8).

Epoxide monomers can also be made, such as by reaction of2-isocyanatoethylmethacrylate with glycidol. Epoxide polymers can alsobe prepared by reacting hydroxyl polymers (e.g.,polyhydroxypropylacrylamide) with epichlorohydrin. Other epoxidemonomers and polymers can be made by those skilled in the art havingspacers of various lengths and with various polarities. Other examplesof suitable monomers are described in U.S. Pat. No. 5,763,629.

Epoxide-containing polymers can also be synthesized by reactinghydroxyl- or amine-containing polymers with diepoxides. Currently, anepoxide activated-Sepharose is available (Sigma) that is made byreacting Sepharose gel beads with 1,4-butanedioldiglycidyl ether. This,or other diepoxides, (e.g., ethylene glycol diglycidyl ether,diepoxyoctane or diepoxydecane) can be used for derivatizing eitheramine or hydroxyl polymers to make polyepoxides. For example,polyhydroxypropylacrylamide or a copolymer containing a photomonomer canbe reacted with an excess of 1,4-butanedioldiglycidyl ether to make apolyepoxide that can then be immobilized onto a surface.

Useful epoxide monomers include those of the general formula:

Where R₁ is either CH₃ or H and X is a noninterfering radical,preferably selected from the group:

where m=2-6 and n=1-10;

where n=1-10;—(CH₂)_(m)-O—(CH₂)—where m=0 or 1, and;

where m=1-20 and n=1-10.

Without intending to be bound by theory, it would appear that epoxidegroups can be used to immobilize underivatized DNA in a covalentfashion, and presumably due to a mechanism of reacting with amine groupson the purine and pyrimidine rings, such as cytosine and adenine, orwith terminal hydroxyl groups.

Reagents of the invention optionally carry one or more pendent latentreactive (preferably photoreactive) groups covalently bonded to thepolymer backbone. Photoreactive groups are defined herein, and preferredgroups are sufficiently stable to be stored under conditions in whichthey retain such properties. See, e.g., U.S. Pat. No. 5,002,582, thedisclosure of which is incorporated herein by reference. Latent reactivegroups can be chosen that are responsive to various portions of theelectromagnetic spectrum, with those responsive to ultraviolet andvisible portions of the spectrum (referred to herein as “photoreactive”)being particularly preferred.

Photoreactive groups respond to specific applied external stimuli toundergo active specie generation with resultant covalent bonding to anadjacent chemical structure, e.g., as provided by the same or adifferent molecule. Photoreactive groups are those groups of atoms in amolecule that retain their covalent bonds unchanged under conditions ofstorage but that, upon activation by an external energy source, formcovalent bonds with other molecules.

The photoreactive groups generate active species such as free radicalsand particularly nitrenes, carbenes, and excited states of ketones uponabsorption of electromagnetic energy. Photoreactive groups may be chosento be responsive to various portions of the electromagnetic spectrum,and photoreactive groups that are responsive to e.g., ultraviolet andvisible portions of the spectrum are preferred and may be referred toherein occasionally as “photochemical group” or “photogroup”.

Photoreactive aryl ketones are preferred, such as acetophenone,benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles(i.e., heterocyclic analogs of anthrone such as those having N, O, or Sin the 10-position), or their substituted (e.g., ring substituted)derivatives. The functional groups of such ketones are preferred sincethey are readily capable of undergoing theactivation/inactivation/reactivation cycle described herein.Benzophenone is a particularly preferred photoreactive moiety, since itis capable of photochemical excitation with the initial formation of anexcited singlet state that undergoes intersystem crossing to the tripletstate. The excited triplet state can insert into carbon-hydrogen bondsby abstraction of a hydrogen atom (from a support surface, for example),thus creating a radical pair. Subsequent collapse of the radical pairleads to formation of a new carbon-carbon bond. If a reactive bond(e.g., carbon-hydrogen) is not available for bonding, the ultravioletlight-induced excitation of the benzophenone group is reversible and themolecule returns to ground state energy level upon removal of the energysource. Photoactivatible aryl ketones such as benzophenone andacetophenone are of particular importance inasmuch as these groups aresubject to multiple reactivation in water and hence provide increasedcoating efficiency. Hence, photoreactive aryl ketones are particularlypreferred.

The azides constitute a preferred class of photoreactive groups andinclude arylazides (C₆R₅N₃) such as phenyl azide and particularly4-fluoro-3-nitrophenyl azide, acyl azides (—CO—N₃) such as benzoyl azideand p-methylbenzoyl azide, azido formates (—O—CO—N₃) such as ethylazidoformate, phenyl azidoformate, sulfonyl azides (—SO₂—N₃) such asbenzenesulfonyl azide, and phosphoryl azides (RO)₂PON₃ such as diphenylphosphoryl azide and diethyl phosphoryl azide. Diazo compoundsconstitute another class of photoreactive groups and includediazoalkanes (—CHN₂) such as diazomethane and diphenyldiazomethane,diazoketones (—CO—CHN₂) such as diazoacetophenone and1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates (—O—CO—CHN₂) suchas t-butyl diazoacetate and phenyl diazoacetate, andbeta-keto-alpha-diazoacetates (—CO—CN₂—CO—O—) such as t-butyl alphadiazoacetoacetate. Other photoreactive groups include the diazirines(—CHN₂) such as 3-trifluoromethyl-3-phenyldiazirine, and ketenes(—CH═C═O) such as ketene and diphenylketene. Photoactivatible arylketones such as benzophenone and acetophenone are of particularimportance inasmuch as these groups are subject to multiple reactivationin water and hence provide increased coating efficiency.

Upon activation of the photoreactive groups, the reagent molecules arecovalently bound to each other and/or to the material surface bycovalent bonds through residues of the photoreactive groups. Exemplaryphotoreactive groups, and their residues upon activation, are shown asfollows.

Photoreactive Group Residue Functionality aryl azides amine R—NH—R′ acylazides amide R—CO—NH—R′ azidoformates carbamate R—O—CO—NH—R′ sulfonylazides sulfonamide R—SO₂—NH—R′ phosphoryl azides phosphoramide(RO)₂PO—NH—R′ diazoalkanes new C—C bond diazoketones new C—C bond andketone diazoacetates new C—C bond and ester beta-keto-alpha- new C—Cbond and diazoacetates beta-ketoester aliphatic azo new C—C bonddiazirines new C—C bond ketenes new C—C bond photoactivated ketones newC—C bond and alcohol

Copolymers can be prepared using epoxide-containing monomers asdescribed above, using techniques known to those skilled in the art.Preferably, the epoxide monomers and comonomers undergo free radicalpolymerization of vinyl groups using azo initiators such as2,2′-azobisisobutyronitrile (AIBN) or peroxides such as benzoylperoxide. The comonomers selected for the polymerization are chosenbased on the nature of the final polymer product. For example, aphotoreactive polymer containing epoxide groups can be prepared by theuse of a monomer mixture that includes one or more monomers containing aphotoreactive group and one or more second monomers containing anepoxide group.

An epoxide-functionalized polymeric reagent of this invention can beprepared by appropriate derivatization of a preformed polymer or, morepreferably, by polymerization of a set of comonomers to give the desiredsubstitution pattern. The latter approach is preferred because of theease of changing the ratio of the various comonomers and by the abilityto control the level of incorporation into the polymer.

The composition of the final polymer can be controlled by mole ratio ofthe monomers charged to the polymerization reaction. Typically theepoxide monomers are used at relatively low mole percentages of thetotal monomer content of the polymerization reaction, with the remainderof the composition consisting of a relatively low mole percent ofphotomonomers, and the remainder of monomers which are neitherphotoreactive nor thermochemically reactive toward the nucleic acidsequence. Examples of such monomers include, but are not limited to,acrylamide, acrylic acid and N-vinylpyrrolidone. Based on the relativereactivities of the monomers used, the distribution of the monomersalong the backbone is largely random.

In a preferred embodiment, for instance, a reagent composition is formedhaving between about 5 mole % and about 25 mole % epoxide monomer (morepreferably between about 10 mole % and about 20 mole %); between about0.1 mole % and about 5 mole % photomonomer (more preferably betweenabout 0.5 mole % and about 2 mole %), and the remainder (to 100 mole %)other monomers.

The present invention provides a method and reagent composition forcovalent attachment of target molecules onto the surface of a substrate,such as slides formed of organosilane-pretreated glass,organosilane-pretreated silicon, silicon hydride, or plastic. In oneembodiment, the method and composition are used to immobilize nucleicacid probes onto plastic materials such as microwell plates, e.g., foruse in hybridization assays. In a preferred embodiment the method andcomposition are adapted for use with substantially flat or moldedsurfaces, such as those provided by organosilane-pretreated glass,organosilane-pretreated silicon, silicon hydride, or plastic (e.g.,polymethylmethacrylate, polystyrene, polycarbonate, polyethylene, orpolypropylene). The reagent composition can then be used to covalentlyattach a target molecule such as a biomolecule (e.g., a nucleic acid)which in turn can be used for specific binding reactions (e.g., tohybridize a nucleic acid to its complementary strand).

Support surfaces can be prepared from a variety of materials, includingbut not limited to plastic materials selected from the group consistingof crystalline thermoplastics (e.g., high and low density polyethylenes,polypropylenes, acetal resins, nylons and thermoplastic polyesters) andamorphous thermoplastics (e.g., polycarbonates and poly(methylmethacrylates). Suitable plastic or glass materials provide a desiredcombination of such properties as rigidity, surface uniformity,resistance to long term deformation, and resistance to thermaldegradation.

The present invention provides a method for immobilizing biomoleculessuch as biopolymers, and particularly those selected from nucleic acids,proteins, polysaccharides, the method comprising the steps of:

a) providing a solid support having a surface,

b) providing a reagent comprising one or more epoxide groups, andoptionally also comprising one or more photogroups,

c) coating the reagent on the support surface (e.g., by dipping,spraying, roll-coating or knife-coating), and covalently attaching thepolymeric reagent to the support surface, e.g., by activation of thephotogroups,

d) providing a biopolymer (e.g., nucleic acid, protein, polysaccharide)having one or more corresponding thermochemical reactive groups (e.g.,amine, hydroxyl, sulfhydryl),

e) attaching the biopolymer to the support by reacting its correspondingreactive group with the bound epoxide group,

f) optionally blocking the remaining unreacted epoxide groups (e.g., bythe use of an amine reagent), and

g) using the resultant coated support surface for its intended purpose,e.g., immobilizing biomolecules, such as nucleic acids for use inhybridization (e.g., on slides for arrays, or in microplate wells),

In a preferred embodiment, the present invention provides a method ofattaching a target molecule to the surface of a substrate, the methodcomprising:

a) providing a reagent composition comprising a polymeric backbonehaving one or more pendent epoxide groups adapted to form covalent bondswith corresponding functional groups on the target molecule,

b) coating and immobilizing the reagent composition on the substratesurface,

c) providing a solution comprising a target molecule having one or morefunctional groups thermochemically reactive with corresponding epoxidegroups provided by the reagent composition,

d) applying an amount (e.g., in the form of discrete small sample volumespots) of the solution on the surface of the substrate surface, and

e) incubating the combination under conditions suitable to permit theepoxide groups provided by the reagent composition to form covalentbonds with corresponding functional groups provided by the targetmolecule in order to attach the target molecule to the surface.

Preferably, for use in preparing and using coated slides, the reagent isadapted to be coated and immobilized on a surface in a manner thatpermits:

-   -   i) a small sample volume of a solution containing the target        molecule to be applied in the form of a discrete spot on the        reagent-coated surface,    -   ii) target molecule present in the sample volume to become        covalently attached to the bound reagent by reaction between its        functional groups and the epoxide groups, and    -   iii) substantially all unattached target molecule to be washed        from the spot without undue detectable amounts of target        molecule in the area surrounding the spot.

In a preferred embodiment, the target molecules are preferably appliedto the epoxide polymer-coated surface in a solution at relatively lowionic strength and slightly alkaline pH (e.g., in 150 mM phosphatebuffer, pH 8.5). Optimal coupling can be achieved by incubating thesurface at high humidity (e.g., 75% relative humidity), which can beachieved by placing the surface within an enclosed storage boxcontaining saturated NaCl at room temperature) for several hours, ormost preferably, overnight. The excess uncoupled epoxide groups are thenblocked with a solution containing 50 mM ethanolamine and 0.1% sodiumdodecyl sulfate (SDS) in 0.1M Tris buffer, pH 9 for 30 minutes at 50° C.The surface is then rinsed with deionized water and washed with4×standard saline citrate (“SSC”)(0.6 M NaCl+0.06 M sodium citrate)containing 0.1% SDS at 50° C. for about 15 to about 60 minutes. Thesurface is then rinsed with deionized water and spun dry in acentrifuge.

When used for preparing microarrays, e.g., to attach capture probes(e.g., oligonucleotides or cDNA) to the microarray surface, such captureprobes are generally delivered to the surface in a volume of less thanabout 1 nanoliter per spot, using printing pins adapted to form thespots into arrays having center to center spacing of about 200 μm toabout 500 μm. Unlike the coupling of DNA from solution and onto thesurface of coated microplate wells, nucleic acids printed in arrays ofextremely small spot sizes tend to dry quickly, thereby altering theparameters affecting the manner in which the nucleic acids contact andcouple with the support. In addition to the design and handling of theprinting pins, other factors can also affect the spot size and/or theultimate hybridization signals, including: salt concentrations, type ofsalts and wetting agents in the printing buffer; hydrophobic/hydrophilicproperties of the surfaces; the size and/or concentration of the nucleicacids; and the drying environments.

In a preferred embodiment, the reagent composition can be used toprepare activated slides having the reagent composition photochemicallyimmobilized thereon. The slides can be stably stored and used at a laterdate to prepare microarrays by immobilizing underivatized oramine-derivatized DNA. The coupling of the capture DNA to the surfacetakes place at pH 8-9 in a humid environment following printing the DNAsolution in the form of small spots.

Activated slides of the present invention are particularly well suitedto replace conventional (e.g., amino-silylated) glass slides in thepreparation of microarrays using manufacturing and processing protocols,reagents and equipment such as micro-spotting robots (e.g., as availablefrom Cartesian), and a micro-spotting device (e.g., as available fromTeleChem International). Suitable spotting equipment and protocols arecommercially available, such as the “ArrayIt”™ ChipMaker 3 spottingdevice. This product is said to represent an advanced version of earliermicro-spotting technology, employing 48 printing pins to deliver as manyas 62,000 samples per solid substrate (e.g., 1 inch by 3 inch standardslide).

The use of such an instrument, in combination with conventional (e.g.,poly-L-lysine coated) slides, is well known in the art. See, forinstance, U.S. Pat. No. 5,087,522 (Brown et al.) “Methods forFabricating Microarrays of Biological Samples”, and the references citedtherein, the disclosures of each of which are incorporated herein byreference.

For instance, the method and system of the present invention can be usedto provide a substrate, such as a coated glass slide, with a surfacehaving one or more microarrays. Each microarray preferably provides atleast about 10/cm² (and preferably at least about 100/cm² distincttarget molecules (e.g., polynucleotide or polypeptide biopolymers). Eachdistinct target molecule 1) is disposed at a separate, defined positionin the array, 2) has a length of at least 10 subunits, 3) is present ina defined amount between about 50 attomoles and about 10 nanomoles, and4) is deposited in selected volume in the volume range of about 0.01nanoliters to about 100 nanoliters. These regions (e.g., discrete spots)within the array can be generally circular in shape, with a typicaldiameter of between about 75 microns and about 1000 microns (andpreferably between about 100 and about 200 microns). The regions arealso preferably separated from other regions in the array by about thesame distance (e.g., center to center spacing of about 100 microns toabout 1000 microns). A plurality of analyte-specific regions can beprovided, such that each region includes a single, and preferablydifferent, analyte specific reagent (“target molecule”).

Those skilled in the art, given the present description, will be able toidentify and select suitable reagents depending on the type of targetmolecule of interest. Target molecules include, but are not limited to,plasmid DNA, cosmid DNA, expressed sequence tags (ESTs), bacteriophageDNA, genomic DNA (includes, but not limited to yeast, viral, bacterial,mammalian, insect), RNA, complementary DNA (cDNA), peptide nucleic acid(PNA), and oligonucleotides.

The invention will be further described with reference to the followingnon-limiting Examples. It will be apparent to those skilled in the artthat many changes can be made in the embodiments described withoutdeparting from the scope of the present invention. Thus the scope of thepresent invention should not be limited to the embodiments described inthis application, but only by embodiments described by the language ofthe claims and the equivalents of those embodiments. Unless otherwiseindicated, all percentages are by weight. Structures of the various“Compounds” identified throughout these Examples can be found in Table 1included below.

EXAMPLE 1 Preparation of 4-Benzoylbenzoyl Chloride (BBA-Cl) (Compound I)

4-Benzoylbenzoic acid (BBA), 1.0 kg (4.42 moles), was added to a dry 5liter Morton flask equipped with reflux condenser and overhead stirrer,followed by the addition of 645 ml (8.84 moles) of thionyl chloride and725 ml of toluene. Dimethylformamide, 3.5 ml, was then added and themixture was heated at reflux for 4 hours. After cooling, the solventswere removed under reduced pressure and the residual thionyl chloridewas removed by three evaporations using 3×500 ml of toluene. The productwas recrystallized from 1:4 toluene:hexane to give 988 g (91% yield)after drying in a vacuum oven. Product melting point pressure and theresidual thionyl chloride was removed by three evaporations using 3×500ml of toluene. The product was recrystallized from 1:4 toluene:hexane togive 988 g (91% yield) after drying in a vacuum oven. Product meltingpoint was 92-94° C. Nuclear magnetic resonance (NMR) analysis at 80 MHz(¹H NMR (CDCl₃)) was consistent with the desired product: aromaticprotons 7.20-8.25 (m, 9H). All chemical shift values are in ppmdownfield from a tetramethylsilane internal standard. The final compoundwas stored for use in the preparation of a monomer used in the synthesisof photoactivatable polymers as described, for instance, in Example 3.

EXAMPLE 2 Preparation of N-(3-Aminopropyl)methacrylamide Hydrochloride(APMA) (Compound II)

A solution of 1,3-diaminopropane, 1910 g (25.77 moles), in 1000 ml ofCH₂Cl₂ was added to a 12 liter Morton flask and cooled on an ice bath. Asolution of t-butyl phenyl carbonate, 1000 g (5.15 moles), in 250 ml ofCH₂Cl₂ was then added dropwise at a rate which kept the reactiontemperature below 15° C. Following the addition, the mixture was warmedto room temperature and stirred 2 hours. The reaction mixture wasdiluted with 900 ml of CH₂Cl₂ and 500 g of ice, followed by the slowaddition of 2500 ml of 2.2 N NaOH. After testing to insure the solutionwas basic, the product was transferred to a separatory funnel and theorganic layer was removed and set aside as extract #1. The aqueous layerwas then extracted three times with 1250 ml of CH₂Cl₂, keeping eachextraction as a separate fraction. The four organic extracts were thenwashed successively with a single 1250 ml portion of 0.6 N NaOHbeginning with fraction #1 and proceeding through fraction #4. This washprocedure was repeated a second time with a fresh 1250 ml portion of 0.6N NaOH. The organic extracts were then combined and dried over Na₂SO₄.Filtration and evaporation of solvent to a constant weight gave 825 g ofN-mono-t-BOC-1,3-diaminopropane which was used without furtherpurification.

A solution of methacrylic anhydride, 806 g (5.23 moles), in 1020 ml ofCHCl₃ was placed in a 12 liter Morton flask equipped with overheadstirrer and cooled on an ice bath. Phenothiazine, 60 mg, was added as aninhibitor, followed by the dropwise addition ofN-mono-t-BOC-1,3-diaminopropane, 825 g (4.73 moles), in 825 ml of CHCl₃.The rate of addition was controlled to keep the reaction temperaturebelow 10° C. at all times. After the addition was complete, the ice bathwas removed and the mixture was left to stir overnight. The product wasdiluted with 2400 ml of water and transferred to a separatory funnel.After thorough mixing, the aqueous layer was removed and the organiclayer was washed with 2400 ml of 2 N NaOH, insuring that the aqueouslayer was basic. The organic layer was then dried over Na₂SO₄ andfiltered to remove drying agent. A portion of the CHCl₃ solvent wasremoved under reduced pressure until the combined weight of the productand solvent was approximately 3000 g. The desired product was thenprecipitated by slow addition of 11.0 liters of hexane to the stirredCHCl₃ solution, followed by overnight storage at 4° C. The product wasisolated by filtration and the solid was rinsed twice with a solventcombination of 900 ml of hexane and 150 ml of CHCl₃. Thorough drying ofthe solid gave 900 g ofN-[N′-(t-butyloxycarbonyl)-3-aminopropyl]-methacrylamide, m.p. 85.8° C.by DSC. Analysis on an NMR spectrometer was consistent with the desiredproduct: ¹H NMR (CDCl₃) amide NH's 6.30-6.80, 4.55-5.10 (m, 2H), vinylprotons 5.65, 5.20 (m, 2H), methylenes adjacent to N 2.90-3.45 (m, 4H),methyl 1.95 (m, 3H), remaining methylene 1.50-1.90 (m, 2H), and t-butyl1.40 (s, 9H).

A 3-neck, 2 liter round bottom flask was equipped with an overheadstirrer and gas sparge tube. Methanol, 700 ml, was added to the flaskand cooled on an ice bath. While stirring, HCl gas was bubbled into thesolvent at a rate of approximately 5 liters/minute for a total of 40minutes. The molarity of the final HCl/MeOH solution was determined tobe 8.5 M by titration with 1 N NaOH using phenolphthalein as anindicator. The N-[N′-(t-butyloxycarbonyl)-3-aminopropyl]methacrylamide,900 g (3.71 moles), was added to a 5 liter Morton flask equipped with anoverhead stirrer and gas outlet adapter, followed by the addition of1150 ml of methanol solvent. Some solids remained in the flask with thissolvent volume. Phenothiazine, 30 mg, was added as an inhibitor,followed by the addition of 655 ml (5.57 moles) of the 8.5 M HCl/MeOHsolution. The solids slowly dissolved with the evolution of gas but thereaction was not exothermic. The mixture was stirred overnight at roomtemperature to insure complete reaction. Any solids were then removed byfiltration and an additional 30 mg of phenothiazine were added. Thesolvent was then stripped under reduced pressure and the resulting solidresidue was azeotroped with 3×1000 ml of isopropanol with evaporationunder reduced pressure. Finally, the product was dissolved in 2000 ml ofrefluxing isopropanol and 4000 ml of ethyl acetate were added slowlywith stirring. The mixture was allowed to cool slowly and was stored at4° C. overnight. Compound II was isolated by filtration and was dried toconstant weight, giving a yield of 630 g with a melting point of 124.7°C. by DSC. Analysis on an NMR spectrometer was consistent with thedesired product: ¹H NMR (D₂O) vinyl protons 5.60, 5.30 (m, 2H),methylene adjacent to amide N 3.30 (t, 2H), methylene adjacent to amineN 2.95 (t, 2H), methyl 1.90 (m, 3H), and remaining methylene 1.65-2.10(m, 2H). The final compound was stored for use in the preparation of amonomer used in the synthesis of photoactivatable polymers as described,for instance, in Example 3.

EXAMPLE 3 Preparation of N-[3-(4-Benzoylbenzamido)propyl]methacrylamide(BBA-APMA) (Compound III)

Compound II 120 g (0.672 moles), prepared according to the generalmethod described in Example 2, was added to a dry 2 liter, three-neckround bottom flask equipped with an overhead stirrer. Phenothiazine,23-25 mg, was added as an inhibitor, followed by 800 ml of chloroform.The suspension was cooled below 10° C. on an ice bath and 172.5 g (0.705moles) of Compound I, prepared according to the general method describedin Example 1, were added as a solid. Triethylamine, 207 ml (1.485moles), in 50 ml of chloroform was then added dropwise over a 1-1.5 hourtime period. The ice bath was removed and stirring at ambienttemperature was continued for 2.5 hours. The product was then washedwith 600 ml of 0.3 N HCl and 2×300 ml of 0.07 N HCl. After drying oversodium sulfate, the chloroform was removed under reduced pressure andthe product was recrystallized twice from 4:1 toluene: chloroform using23-25 mg of phenothiazine in each recrystallization to preventpolymerization. Typical yields of Compound III were 90% with a meltingpoint of 147-151° C. Analysis on an NMR spectrometer was consistent withthe desired product: ¹H NMR (CDCl₃) aromatic protons 7.20-7.95 (m, 9H),amide NH 6.55 (broad t, 1H), vinyl protons 5.65, 5.25 (m, 2H),methylenes adjacent to amide N's 3.20-3.60 (m, 4H), methyl 1.95 (s, 3H),and remaining methylene 1.50-2.00 (m, 2H). The final compound was storedfor use in the synthesis of photoactivatable polymers as described, forinstance, in Examples 5 and 6.

EXAMPLE 4 Synthesis of a Spaced Epoxide Monomer (Compound IV)

To three ml of chloroform was added isocyanatoethylmethacrylate (1.0 ml,7.04 mmole), glycidol (0.50 ml, 7.51 mmole) and triethylamine (50 μl,0.27 mmole). The reaction was stirred at room temperature overnight. Theproduct was purified on a silica gel column and the structure confirmedby NMR. The yield was 293 mg (18% yield).

EXAMPLE 5 Synthesis of a Copolymer of Acrylamide, BBA-APMA and SpacedEpoxide Monomer (Compound V)

Acrylamide (1.12 gm, 15.7 mmoles), BBA-APMA (30 mg, 0.085 mmole) andspaced epoxide monomer (Compound IV) (273 μl, 1.28 mmole) were dissolvedin 15.6 ml of tetrahydrofuran (THF). To this solution was added 34 mg of2,2′-azobisisobutyronitrile (AIBN) and 16 μl ofN,N,N′,N′-tetramethylethylenediamine (TEMED). The solution was spargedwith helium for four minutes, then argon added to head space, thentightly capped and placed in a 55° C. oven overnight. The reactionmixture containing precipitated polymer was centrifuged and thesupernatant decanted. The residue was resuspended in 20 ml of fresh THF,centrifuged and decanted. This was repeated, followed by filtering andfurther washing of the polymer with two ten ml aliquots of THF. Thepolymer was the dried under vacuum to a constant weight. The yield was1.477 gm.

EXAMPLE 6 Preparation of Copolymer of Acrylamide, BBA-APMA, and GlycidylMethacrylate (Photo PA-Polyepoxide) (Compound VI)

Acrylamide (7.1 gm, 99.35 mmoles), BBA-APMA (0.414 gm, 1.18 mmole) and2,2′-azobis(2-methylbutyronitrile) (“VAZO 67”, 0.262 gm, 1.4 mmole) weredissolved in 108 ml of THF. To this solution was added 2.4 ml ofglycidylmethacrylate (17.7 mmoles). The solution was sparged with heliumfor four minutes, then with argon for four minutes, then capped tightlyand heated at 61° C. overnight while mixing. The polymer was collectedby filtration, then suspended in methanol and mixed, after which it wasagain collected by filtration and washed with chloroform, then dried ina vacuum oven at 30° C.

EXAMPLE 7 Preparation of Microscope Slides Coated with PolyEpoxide

Soda lime glass microscope slides (Erie Scientific, Portsmouth, N.H.)were silane treated by dipping in a mixture ofp-tolyldimethylchlorosilane (T-Silane) and N-decyldimethylchlorosilane(D-Silane, United Chemical Technologies, Bristol, Pa.), 1% each inacetone, for 1 minute. After air drying, the slides were cured in anoven at 120° C. for one hour. The slides were then washed with acetonefollowed by DI water dipping. The slides were further dried in an ovenfor 5-10 minutes.

Compound VI was sprayed onto the silane treated slides, which were thenilluminated using a Dymax lamp (25 mjoule/cm² as measured at 335 nm witha 10 nm band pass filter on an International Light radiometer) whilewet, washed with water, and dried.

EXAMPLE 8 Preparation and Use of Microarrays with PhotoPA-PolyepoxideCoated Slides

PCR products derived from actin, glucose phosphate dehydrogenase (GPDH)or β-galactosidase genes were printed onto slides at 0.1 mg/ml in 0.15 Mphosphate buffer using an X, Y, Z motion controller to positionChipMaker 2 microarray spotting pins (Telechem International). Theslides were either coated with Photo-PA-polyepoxide as in Example 7 orpolylysine slides prepared by published methods (See U.S. Pat. No.5,087,522 (Brown et al.) “Methods for Fabricating Microarrays ofBiological Samples”, and the references cited therein). The printedepoxide slides were incubated overnight at room temperature and 75%relative humidity. The printed polylysine slides were UV crosslinked.After printing, the epoxide slides were blocked with 50 mM ethanolaminein 0.1M tris buffer, pH 9.0 and washed. The polylysine slides wereprocessed by the published procedure. Both types of slides werehybridized with Cy5-labeled total RNA either spiked with β-gal mRNA at1:250,000 or not spiked. The slides were then scanned with a laserscanner to measure intensities of Cy5 fluorescence.

No β-Galactosidase Spike β-Galactosidase Spike Actin GPDH β-Gal ActinGPDH β-Gal Epoxide 33913 ± 5984 ± 211 ± 47351 ± 29007 ± 9571 ± Coating 4370  443  27  2170  8825 2531 Poly-L- 29946 ± 4751 ± 282 ± 34836 ±11545 ± 4524 ± Lysine  1805  686  32  2222  1882  825

EXAMPLE 9 Preparation and Use of Microarrays with Photo-polyepoxideCoated Slides

Oligonucleotides, either aminated or nonaminated, were printed ontoslides at 8 μM in 0.15 M phosphate buffer using an X, Y, Z motioncontroller to position ChipMaker 2 microarray spotting pins (TelechemInternational). The slides were either coated with photo-PA-polyepoxideas in Example 7, with photo-PA-PolyNOS by the same procedure or withpolylysine by published methods (See references in Example 8). Theprinted epoxide and NOS slides were incubated overnight at roomtermperature and 75% relative humidity. The printed polylysine slideswere processed by the published procedure. The slides were scanned tomeasure the Cy3 fluorescence of immobilized capture oligos, thenhybridized at 41° C. overnight with Cy5-labeled oligonucleotide (1pmole/slide) that was complementary to the capture oligos. The slideswere scanned with a laser scanner to measure the fluorescenceintensities of the hybridized oligos. Because the amount of captureoligo immobilized with the amine-silane slides was so low, they were nothybridized.

Capture Oligo Hybridization Immobilized Signal Coated Amine- Non-amine-Amine- Non-amine- Slides oligo oligo oligo oligo NOS 30,302 ± 5037 ±25,226 ± 6116 6090 ± 503 3866 294 Epoxide 36,793 ± 30,821 ± 26,526 ±10887 28,467 ± 11695 5145 3585 Amino- 800 ± 492 ± ND ND silane 85 92

TABLE 1 Compounds

COMPOUND I

COMPOUND II

COMPOUND III

COMPOUND IV

COMPOUND V (where x = 0.1 to 5 mole %, y = 2 to 30 mole % and z = 65 to97.9 mole %)

COMPOUND VI (where x = 0.1 to 5 mole %, y = 2 to 30 mole % and z = 65 to97.9 mole %)

1. An activated slide comprising a flat support surface coated with areagent composition for attaching a target molecule to the surface;wherein the reagent composition comprises a polymeric backbone, thepolymeric backbone having one or more pendent photoreactive groups andone or more pendent epoxide groups attached thereto; wherein the reagentcomposition is covalently attached to the surface via activation of thependent photoreactive groups; and wherein the one or more pendentepoxide groups are capable of forming covalent bonds with the targetmolecules.
 2. An activated slide according to claim 1 wherein the slideprovides a surface for fabricating a microarray wherein the targetmolecule comprises a nucleic acid and the surface comprisesorganosilane-pretreated glass, organosilane-pretreated silicon, siliconhydride, or plastic.
 3. An activated slide according to claim 1 whereinthe slide provides a microarray with at least about 10/cm² nucleic acidshaving a length of at least 10 nucleotides, the nucleic acids each beingspotted in discrete regions and defined amounts of between about 50attomoles and about 10 nanomoles.
 4. An activated slide according toclaim 3 wherein the regions are generally circular in shape, the regionshaving a diameter of between about 75 microns and about 1000 microns andseparated from other regions in the microarray by center to centerspacing of about 100 microns to about 1000 microns.
 5. An activatedslide according to claim 1 wherein the target molecule is a nucleic acidand the photoreactive group comprises photoreactive aryl ketone.
 6. Anactivated slide according to claim 5 wherein the photoreactive arylketone is selected from the group consisting of acetophenone,benzophenone, anthroquinone, anthrone, and anthrone-like heterocycles.